The present invention relates generally to the separation and analysis of complex materials, specifically biological materials. More particularly, the present invention relates to methods for the multiplexed separation and/or characterization of components of complex biological mixtures utilizing solid phase extraction techniques, preferably on a micro- or nanoscale. In some preferred embodiments, the present invention employs combinatorially derived extraction phases on nanoparticles to extract analytes from a sample.
A variety of methods have been developed for the separation of mixtures for analysis (e.g., filtration, chromatography, extraction, electrophoresis, etc.). However, these methods have not proven sufficient for the separation of biological samples (e.g., blood, plasma, serum, synovial fluid, cerebrospinal fluid, saliva, tears, bronchial lavages, urine, stool, excised organ tissue, bone marrow, etc.). Such samples are comprised of a complex and heterogeneous mixture of molecular and cellular material in which certain components may be quite abundant, while others are present in only trace amounts. The separation and analysis of these types of samples have presented challenges to scientists using conventional techniques.
For example, the currently preferred method of performing proteome analysis (xe2x80x9cproteomicsxe2x80x9d) uses two-dimensional (2-D) gel electrophoresis to separate complex protein mixtures. After electrophoresis and staining, the revealed spots of the gel are excised. The protein is then separated from the gel and subjected to enzymatic digestion. The resulting peptide fragments are then typically characterized by mass spectrometry, such as Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF MS) or electrospray ionization (ESI-MS). The original protein structure is then reconstructed by matching the peptide masses against theoretical peptide masses for known proteins that can be found in protein sequence databases, such as SWISS-PROT. Shortcomings of this technology include the lack of reproducibility of the 2-D gel process, difficulties in protein quantitation, and sample loss when recovering the protein from the gel. 2-D gels also suffer from a separation bias against proteins (and other molecules) of very low and very high molecular weight, and against proteins with the same pI. Accordingly, 2-D gels cannot be used for profiling small organic molecules, chemokines, metabolites, and so on. Many molecules known to be important in various disease states (e.g., cholesterol, thyroid hormone, etc.) are, therefore, not detectable by this method.
Specific affinity binding is an additional technique used to capture specific target ligands from complex mixtures such as biological fluids. For example, monoclonal or polyclonal antibodies, may be immobilized on a surface. When the surface is contacted with the sample, the antibodies bind to components of the mixture. Analysis can be carried out via competitive binding, or in sandwich mode using a secondary antibody. In both modes, there is typically a tag (enzyme, radiolabel, fluorophore, etc.) that is used for detection and/or amplification. Specific affinity binding techniques have been applied to proteomics in order to characterize gene products. Although it is highly specific, such immunoseparation has many of the same drawbacks as other assays that take place in two dimensions. Moreover, immunoseparation fails when there is no high-affinity antibody available to components in the sample, which is often the case. In particular, immunoseparation fails for (i) unknown molecules, (ii) known protein molecules that are post-translationally modified at or near the high affinity epitope; and (iii) molecules too small to elicit a strong immune response.
One specific affinity binding approach to proteomics where the analysis is limited to known proteins (i.e., proteins for which antibodies are commercially available), is the state-of-the-art FlowMetrix system developed and commercialized by Luminex Corp. (Austin, Tex.). The FlowMetrix system uses microspheres as the solid support for performing multiplexed immunoassays. Currently Luminex offers 64 different bead sets. Each bead set can, in principle, support a separate immunoassay and the beads are read using an instrument similar to a conventional flow cytometer. A major limitation of the Luminex approach is that the frequency space of molecular fluorescence used both for microsphere tagging and detection is not wide enough to accommodate nearly as many different assays as would be desirable to fully realize the advantages of multiplexing.
Solid phase microextraction (SPME) is a solvent-free separation technique that combines sampling and analyte concentration. The basic process of solid phase extraction involves adsorption of one or more target analytes from a sample matrix into a solid xe2x80x9cextractionxe2x80x9d phase. During the extraction, exposure of the extraction phase to the sample leads to the partitioning of analyte between the sample and extraction phases. The amount that any particular analyte is extracted from the sample depends on a number of factors, including the partition coefficient.
A device for performing SPME was the subject of U.S. Pat. No. 5,691,206, entitled, xe2x80x9cMethod and Device for Solid Phase Microextraction and Desorption.xe2x80x9d As described therein, a thin coat of polymer or other extraction phase is coated on a fused silica fiber. The coated fiber, or probe, is contained within a hollow needle extending from the barrel of a syringe-like apparatus and can be extended or retracted using a plunger. To extract analytes from a sample, the needle is inserted into the sample and the coated fiber is extended into the sample. The sample matrix containing the analytes can be a gaseous sample, a liquid sample, or even the headspace above a liquid sample. After the microextraction has been allowed to take place, the fiber is retracted and the needle is removed from the sample.
The extracted analytes can then be delivered to a suitable instrument for analysis. SPME has been successfully coupled to high pressure liquid chromatography (HPLC) and gas chromatography (GC). For analysis by mass spectrometry (MS), analytes adsorbed into the extraction phase may be thermally desorbed and studied by MALDI-MS or Surface Assisted Laser Desorption Ionization mass spectrometry (SALDI-MS), or the analytes may be ionized by electrospray techniques.
SPME has been used for numerous applications in pharmaceutical science, environmental science, biological science, and chemical science. In short, SPME can be used for any application in which chromatographic separation is desired. In many contexts, SPME is simpler, faster and produces extracts of greater purity than traditional solvent-solvent extractions. SPME has been successfully used, for example, to extract pyrazines from peanut butter, fatty acids from milk, and amphetamines from biological fluids.
As it is currently practiced, SPME has several important limitations. First, performing SPME using a single fiber does not allow for multiplexing. The single needle method described in the literature would be of limited value for larger scale efforts that require many experiments to be run simultaneously in the same sample. It would be impracticable, for example, for a full-scale proteomics effort to rely on existing SPME.
Second, the limited number of solid extraction phases currently available necessarily limits SPME""s selectivity as a separation technique. In the original SPME literature, the extraction phase associated with the fiber probe was polydimethylsiloxane (PDMS) or polyacrylamide (PA). These materials possess the fundamental properties necessary to effect SPMExe2x80x94they are chemically stable; are able to be cast as a thin film; have a semi-porous or porous geometry, and have a reasonably high affinity for one or more classes of molecules. In particular, PDMS has a high affinity for non-polar organics and PA has a high affinity for polar organics. However, neither material exhibits particularly high affinity for water-soluble species. Efforts to address the limited selectivity of SPME extraction phases have met with only limited successxe2x80x94there are now roughly ten different commercially available extraction phases for use in SPME. However, considering the diversity of structure present in the proteome, as well as in the roughly 10,000 different low molecular weight species known to be present in blood, it is clear that SPME in its current method of practicexe2x80x94using single needle extractions and a small number of different extraction phasesxe2x80x94is of limited utility for comprehensive profiling of biological samples.
In some cases, researchers have resorted to using two or more different separation methods in order to profile complex mixtures. However, such xe2x80x9chyphenated separation techniquesxe2x80x9d generally require increased sample volume and have been hampered by incompatibilities with respect to different separation techniques and the methods eventually used to analyze the separated analytes.
Superimposed on the challenges presented using conventional techniques to analyze biological samples, is the pressure to do so faster and with smaller sample sizes. Indeed, advances in medicine and biology have resulted in a paradigm change in what is traditionally defined as bioanalytical chemistry. A major focus of new technologies is to generate what could be called xe2x80x9cincreased per volume information content.xe2x80x9d This term encompasses several approaches, from reduction in the volume of sample required to carry out an assay, to highly parallel measurements (xe2x80x9cmultiplexingxe2x80x9d), such as those involving immobilized molecular arrays, to incorporation of second (or third) information channels, such as in 2-D gel electrophoresis or CE-electrospray MS/MS. It also encompasses efforts to achieve miniaturization of the machinery of analysisxe2x80x94as in Bio-Microelectromechanical systems (Bio-MEMS), microfabricated devices using silicon, glass and polymer substrates that have been utilized in electrophoresis, electrochemistry and chromatography to reduce sample volume and increase speed and throughput. (See, e.g., Manz, A., Becker, H. Eds., xe2x80x9cMicrosystem Technology in Chemistry and Life Science,xe2x80x9d Springer-Verlag: Berlin (1998)).
Unfortunately, many of these seemingly revolutionary technologies are limited by a reliance on relatively pedestrian materials, methods, and analyses. For example, the development of DNA microarrays (xe2x80x9cgene chipsxe2x80x9d) for analysis of gene expression and genotyping by Affymetrix, Inc., Incyte Genomics and others provides the wherewithal to immobilize up to 20,000 different fragments or full-length pieces of DNA in a spatially-defined 1 cm2 array. At the same time, however, the use of these chips in all cases requires hybridization of DNA in solution to DNA immobilized on a planar surface, which is marked both by a low efficiency of hybridization (especially for cDNA) and a high degree of non-specific binding. It is unclear whether these problems can be completely overcome. Moreover, there is a general sense of disillusionment both about the cost of acquiring external technology and the lead-time required to develop DNA arraying internally.
A second example of how groundbreaking techniques can be slowed by inferior tools, is in pharmaceutical discovery by combinatorial chemistry. Solution phase, 5-10 xcexcm diameter latex beads are used as sites for molecular immobilization in some protocols. Exploiting the widely adopted xe2x80x9csplit and poolxe2x80x9d strategy, libraries of upwards of 100,000 compounds can be simply and rapidly generated. As a result, the bottleneck in drug discovery has shifted from the synthesis of candidates to screening, and equally importantly, to compound identification, (i.e., knowing which compound is on which bead). Current approaches to the latter problem include xe2x80x9cbead encodingxe2x80x9d, whereby each synthetic step applied to a bead is recorded by the parallel addition of an organic xe2x80x9ccodexe2x80x9d molecule. Reading the code allows the identity of the drug lead on the bead to be identified. Unfortunately, the xe2x80x9ccode readingxe2x80x9d protocols are far from optimal. In such strategies, the code molecule must be cleaved from the bead and separately analyzed by HPLC, mass spectrometry or other methods. In other words, there is at present no way to identify potentially interesting drug candidates by direct, rapid interrogation of the beads on which they reside, even though there are numerous screening protocols in which such a capability would be desirable.
Two alternative technologies with potential relevance both to combinatorial chemistry and genetic analysis involve xe2x80x9cself-encoded beadsxe2x80x9d, in which a spectrally identifiable bead substitutes for a spatially defined position on a solid supporting chip. In the approach pioneered by Walt and co-workers, beads are chemically modified with a ratio of fluorescent dyes intended to uniquely identify the beads, which are then further modified with a unique chemistry (e.g., a different antibody or enzyme). The beads are then randomly dispersed on an etched fiber array so that one bead associates with each fiber. The identity of the bead is ascertained by its fluorescence readout, and analytes are detected by fluorescence readout at the same fiber in a different spectral region. The seminal reference (Michael et al., Anal. Chem., 22, 1242-1248 (1998)) describing this technology suggests that with 6 different dyes (15 combinations of pairs) and with 10 different ratios of dyes, 150 xe2x80x9cunique optical signaturesxe2x80x9d could be generated, each representing a different bead xe2x80x9cflavorxe2x80x9d. A very similar strategy is used by at Luminex, that combines flavored beads ready for chemical modification (100 commercially available) with a flow cytometry-like analysis. (See, e.g., McDade et al., Med. Rev. Diag. Indust., 19, 75-82 (1997)). Once again, the particle flavor is determined by fluorescence, and once the biochemistry is put onto the bead, any spectrally distinct fluorescence generated due to the presence of analyte can be detected. Note that as currently configured, it is necessary to use one color of laser to interrogate the particle flavor, and another, separate laser to excite the bioassay fluorophores.
A significant limitation of self-encoded latex beads is that imposed by the wide bandwidth associated with molecular fluorescence. If the frequency space of molecular fluorescence is used both for encoding and for bioassay analysis, it is hard to imagine how, for example, up to 20,000 different flavors could be generated. This problem may be alleviated somewhat by the use of combinations of glass-coated quantum dots, which exhibit narrower fluorescence bandwidths. (See, e.g., Bruchez et al., Science, 281, 2013-2016 (1998)). If, however, it were possible to generate very large numbers of intrinsically-differentiable particles by some means, then particle-based bioanalysis would become exceptionally attractive, insofar as a single technology platform could then be considered for the multiple high-information content research areas; including combinatorial chemistry, genomics, and proteomics (via multiplexed immunoassays).
Surface derivatized probes consisting of self-assembled monolayers (SAMs) terminated with ionic functional groups also have been used for extracting peptides/proteins. (Warren et al., Anal. Chem., 70, 3757-3761 (1998)).
SPME followed by CE as the second dimension has been used to analyze a mixture of peptides from a proteolytic digest. (Tong et al., Anal. Chem., 71, 2270-2278 (1999)). Although the SPME-CE/MS improved the concentration detection limit by more than two orders of magnitude when compared to CE-MS alone, the large electro-osmotic force of the aminopropylsilane (APS) coated capillary tended to elute all the peptides in a relatively short period of time. This presents the possibility of confounding results owing to the co-elution of compounds.
A strategy has been used for the separation of MHC class I peptides, several thousand peptides at sub-femtomolar concentrations. The literature reports immuno-affinity concentration followed by reverse phase, and subsequently concentrated on specially designed membranes capillaries. (Tomlinson et al., J. Caromator. A, 744, 237-78 (1996)). In addition, a comprehensive two-dimensional separation technique has been described for profiling proteins. (Opiteck et al., Anal. Chem., 62, 1518-1524 (1997)).
There is a need for analytical methods of high sensitivity and selectivity that have the power to resolve and profile different components of a complex mixture, such as a biological fluid. At the same time, there is a need for such methods to be able to identify and preferably quantitate minute quantities of biomolecules in small sample sizes, potentially even in single cells.
There is also a need for streamlined and automated methods for analyte capture that are compatible with sophisticated separation and detection technologies, such as HPLC, GC, CE, and MS.
There is also a need for methods of rapidly interrogating a biological sample that can be multiplexed. In particular, there is a need to have methods for separation and analysis of low-molecular weight organic molecules, peptides, and larger proteins simultaneously in a microvolume samples.
There is a need for combinatorially-derived extraction phases to extract analytes from a sample. In particular, there is a need for such surfaces that can be used in multiplexed analyses.
The present invention relates generally to methods for multiplexed separation and analysis of biological materials. More particularly, the present invention relates to methods for multiplexed characterization of components of biological materials utilizing solid phase extraction techniques, preferably on a micro- or nanoscale. The solid phase extraction methods of the present invention are accomplished using solid supports that have been coated or are otherwise associated with an extraction phase. In some preferred embodiments, the present invention employs combinatorially derived extraction phases on nanoparticle supports. In other preferred embodiments, the solid supports for the extraction phases are arrays of fibers. In some preferred embodiments, the present invention relates to methods and materials for performing solid phase extraction and analysis using nanoparticles coated with an extraction phase to extract analytes from a sample.
The resent invention includes a method for performing solid phase extraction using particles as the stationary support or probe. In preferred embodiments, such particles are differentiable from each other. In some preferred embodiments, the particles are nanobarcodes which allow extremely high-level assay multiplexing in solution, essentially combining the advantages of arrays (e.g., gene and/or protein chips) with the advantages of solution-based assays. Nanobarcodes may be used according to the present invention to simultaneously perform, for example, thousands of chemically and biochemically selective nanoscale extractions on samples, and then analyzing the extracted molecules, including by using mass spectrometry and/or fluorescence. Although not necessary to achieve the benefits of the present invention, the use of nanobarcode technology increases the power of the analytical separation methods described herein.
In some preferred embodiments, the present invention includes methods of carrying out solid-phase nanoextractions (SPNE). Such methods preferably employ particles that are distinguishable from one another. Other solid supports included within the scope of the invention are beads. Also included within the scope of the invention are methods for the simultaneous use of a plurality of differentiable solid supports, each associated with a different extraction phase for solid phase nanoextraction.